Molecular hydrogen present in water in a dissolved form (dissolved hydrogen) is an important indicator of various biological and chemical processes. These processes include in situ bioremrediation of groundwater by engineered methods or by natural attenuation, anaerobic reactors for waste treatment including anaerobic digesters, anaerobic bioprocesses for the manufacture of biochemicals, including fermentation, operation of subsurface, permeable metal-reactive walls for remediation of chlorinated chemicals in groundwater by reductive dehalogenation and corrosion of metals in process systems including boilers. Dissolved hydrogen can be an indicator of the nature, extent, or stability of these processes.
The concentration of dissolved hydrogen can be extremely low. For example, one class of anaerobic bacteria known as iron-reducing bacteria typically demonstrate dissolved hydrogen concentrations in groundwater in the range of 0.1 to 1.0 nM when at steady state (F. H. Chapelle and P. B. McMahon, J. Hydrology, 127:85-108 (1991)).
Methods available for measurement of dissolved hydrogen involve direct measurement in the liquid of interest or extraction of dissolved hydrogen into a carrier gas which is then analyzed. Only one method exists for measuring dissolved hydrogen concentrations as low as 0.1 nM and is called the "bubble strip" method (F. H. Chapelle and P. B. McMahon, J. Hydrology, 127:85-108 (1991)). This method involves equilibration of a bubble of nitrogen with a flowing stream of groundwater in a gas sampling bulb made from glass. Samples of the gas bubble are injected into a reduction gas analyzer over time until the gas bubble is in equilibrium with the groundwater. The reduction gas analyzer employs chemical reduction of a heated bed of mercuric oxide by hydrogen to form gaseous mercury that is sensed by an ultraviolet detector. Chromatographic separation of hydrogen from other reducing gases is required prior to mercuric oxide reduction. The gaseous hydrogen concentration is then related to the dissolved hydrogen concentration by Henry's law where 0.1 nM dissolved hydrogen approximately correlates to 0.125 ppm of gaseous hydrogen at equilibrium and ambient temperature and pressure. This method is difficult, time-consuming, and expensive to use and has therefore not gained widespread acceptance as an analytical method. Application of the reduction gas analyzer, in combination with hydrogen equilibration over Teflon tubing, to anaerobic digestion in particular is cited as being limited because of its "sophistication, high cost, detection limits, and interference from other solutes" (K. Kuroda, R. G. Silveira, N. Nishio. H. Sunahara, and S. Nagai, J. Ferment. Boeing, 71:418-423 (1991)). The gas bubble equilibration method also is greatly subject to operator error, in part because of mass transfer limitations. A. Pauss, G. Andre, M. Perrier, and S. R. Guiot, Appl. Environ Microbiol, 56:1636-1644 (1990). Other methods that are available for hydrogen measurement are insensitive at these low concentrations and in environments of interest.
A Clark probe with reversed polarity is capable of hydrogen measurement in gases or liquids. The lower detection limit is 500 ppm in gases (F. J. Hanus, K. R. Carter, and H. J. Evans, Methods in Enzymology, 69:731-739 (1980)) and 15 .mu.g/L (7,500 nM) in water (J. D. Istok, M. D. Humphrey, M. H. Schroth, M. R. Hyman, and K. T. O'Reilly, Ground Water, 35:619-631 (1997)). Another electrochemical probe for dissolved hydrogen described by Strong (G. E. Strong and R. Cord-Ruwisch, BiotechnoL Boeing, 45:63-68 (1995)) has a detection limit of 30 Pa partial pressure which is equivalent to 240 nM. Ozawa et al. in EP 0096417A1 describe an electrochemical hydrogen sensor that has a sensitivity of 500 nM dissolved hydrogen. Kitamura et al. in EP 0122511 A2 describe a similar electrochemical hydrogen sensor that compensates for oxygen but does not remove its influence and has an insufficient sensitivity in the nM range. Other electrochemical methods employing fuel cells have been described with detection limits of 1 .mu.M (1,000 nM) (J.-P. Gebeault, J. Van Berlo, and M. Dymarski, Trans. Amer. Nucl Soc., 46:612-613 (1984)) and 80 nM. A. Pauss, R. Samson, S. Guiot and C. Beauchemin, Biotechnol. Bioeng., 35:492-501 (1990). Hydrogen sulfide and oxygen interfere with the performance of these probes. A. Pauss, R. Samson, S. Guiot and C. Beaucherun, Biotechnol. Bioeng., 35:492-501 (1990). In one case, oxygen did not interfere as long as it was present in lower concentrations than hydrogen (N. Hara and D. D. Macdonald, J. Electrochem. Soc., 144:4152-4157 (1997)). In the practice of the present invention, very low hydrogen concentrations render this requirement impractical.
Gas chromatography with thermal conductivity detection can be used to detect hydrogen in gases. This method can be used to detect 0.5 nmoles of injected hydrogen (F. J. Hanus, K. R. Carter, and H. J. Evans, Methods in Enzymology, 69:731-739 (1980)) which, based on a 1-ml injection, translates to a concentration of 12 ppm in gas or an equilibrium dissolved concentration of 9.6 nM.
An instrument based on thermal conductivity has been developed to measure hydrogen in steam or hydrogen dissolved in water and has an inadequate detection limit of 100 nM (C. R. Wilson, Electric Power Research Institute Report NP-2650 (1982)).
Equilibration of dissolved hydrogen in water with a carrier gas followed by removal of coexisting gases (e.g., oxygen, hydrogen sulfide, carbon dioxide) that can interfere with or dilute hydrogen during analysis has been attempted but not at sufficiently low detection limits. Removal of carbon dioxide from carrier gas equilibrated with rumen fluid followed by gas chromatography resulted in a detection limit of 10 nM (J. A. Robinson, R. F. Strayer, and J. M. Tiedje, Appl. Environ. Microbiol, 41:545-548 (1981)). This method is not applicable where carbon dioxide is present in low concentrations.
Mass spectrometry can be used to detect hydrogen in gases or, via use of a membrane system, in liquids (P. Dornseiffer, B. Meyer, and E. Heinzle, Biotechnol. Bioeng., 45:219-228 (1995)). Hydrogen concentrations detected in liquids are in the low .mu.M (1,000 nM) range and accurate measurement can be compromised by biofilm growth on the membrane surface which requires periodic maintenance and cleaning.
A palladium-coated micromirror fiber optic sensor developed by Sandia National Laboratories was shown to be capable of sensing 50 ppm of hydrogen in transformer oil (M. A. Butler, R. Sanchez, and G. R. Dulleck, Sandia Report SAND96-1133. UC-706 (1996)).
Various types of solid state sensors are capable of hydrogen detection. eithley (Cleveland, Ohio) sells a hot wire semiconductor type sensor named CH-H. This sensor contains a platinum wire in a sintered tin oxide semiconductor bead. Hydrogen reacts with oxygen on the platinum wire thereby generating heat. The altered resistance of the platinum wire is sensed in a bridge circuit. This sensor requires the presence of oxygen and is sensitive to approximately 10 ppm hydrogen in gas or an equilibrium dissolved concentration of 8 nM.
Sensors based on the observed change in the electrical resistance of platinum and palladium upon adsorption of hydrogen have been described. These sensors can be immersed in water but have a detection limit of 5,000 nM dissolved hydrogen (C. Liu and D. D. Macdonald, J. Supercritical Fluids., 8:263-270 (1995)).
Lundstrom described metal oxide semiconductor (MOS) transistors containing a palladium gate (K. I. Lundstrom, M. S. Shivaraman, and C. M. Svensson, J. Appl. Physics., 46:3876-3880 (1975); I. Lundstrom, Sensors and Actuators., 1:403-426 (1981)). The sensitivity of these structures to hydrogen in gas is highly dependent on oxygen concentration. A 10 mV response was observed with 0.5 ppm hydrogen in air and with 0.03 ppb hydrogen in an inert gas such as argon or nitrogen. The difference in response is due to the oxygen content of air. These sensors are also sensitive to hydrogen sulfide albeit at ten-fold greater concentrations than hydrogen (I. Lundstrom, Sensors and Actuators., 1:403-426 (1981)) and sulfur compounds are well known for their poisoning of metallic surfaces. A hydrogen leak detector based on such MOS sensors demonstrated a practical sensitivity of 1 ppm (L. Stiblert and C. Svensson, Rev. Sci. Instrum., 46:1206-1208 (1975)).
A hydrogen sensor with a practical sensitivity of 1 ppm in gas is described by Hughes et al. in U.S. Pat. No. 5,279,795. This type of sensor is disadvantageous in part because of the slow response at low hydrogen concentrations. The sensitivity of this sensor is negatively affected by the presence of oxygen. It was reported that hydrogen sulfide does not poison the sensor; however, the tests were conducted in air where hydrogen sulfide poisoning is known to be mitigated by oxidation. This sensor has been incorporated into a hand held detector by DCH Technology which has a detection limit of 10 ppm in gas or an equilibrium dissolved concentration of 8nM.
Immersion of MOS devices in anaerobic water is not practical because of incompatibility. Protection of a MOS device with a gas-permeable membrane such as Goretex.TM. would be expected to work for detection of dissolved hydrogen in anaerobic water but does not. While anaerobic conditions in groundwater would seem to imply the absence of oxygen; in fact, oxygen is often observed in "anaerobic" groundwater, presumably due to the heterogeneous nature of many aquifers. Additionally, MOS devices are poisoned by hydrogen sulfide. Hydrogen sulfide is a common contaminant present in anaerobic groundwater and in anaerobic digesters. These sensors are also inhibited by carbon monoxide which is found in anaerobic environments.
Neuwelt in U.S. Pat. No. 3,661,010 describes a method employing an electrochemical sensor covered by a membrane over which flows the liquid. This method is disadvantageous because no method for removal of interferences is provided and insufficient sensitivity exists. A dissolved hydrogen analyzer manufactured by Orbisphere Laboratories (Inverness, Calif.) also uses an electrochemical sensor covered by a membrane but is sensitive only to 15 nM dissolved hydrogen and this sensitivity is adversely affected by oxygen.
Immersion of any type of hydrogen probe in a biological medium can also result in growth of biofihm on the probe. Such biofilm growth can subsequently result in dissolved hydrogen consumption or production which can affect the measurement accuracy. Such effects were observed with a gas diffusion probe used in conjunction with a reduction gas analyzer (H. Kramer and R. Conrad, FEMS Microbiol. Ecol., 12:149-158 (1993)).
Schuy in U.S. Pat. No. 3,920,396 describes a membrane equilibration device that uses an extraction gas circulating in a closed loop to attain equilibrium between the gas and liquid sample of fixed volume. This method is disadvantageous because dissolved gases with high Henry constants will be predominately stripped into the gas phase, and the attained equilibrium will occur at a dissolved gas concentration that is significantly less than the original dissolved gas concentration. Furthermore, this method provides no means for removal of interfering gases that also equilibrate across the membrane.
Baillie et al. in U.S. Pat. No. 4,916,079 describe a gas-liquid equilibration device that uses a constant flow of liquid which overcomes the disadvantages of U.S. Pat. No. 3,920,396 by using a continuous flow of liquid and spiking the equilibration gas with a known quantity of the analyte to overcome interferences. This method is not applicable to the analysis of low levels of hydrogen in the practice of the present invention because the concentrations of hydrogen are too low relative to the concentrations of interfering gases.
Ketchum et al. in U.S. Pat. No. 4,236,404 describe a device to monitor hydrogen and other gases in electrical insulating liquids such as transformer oils that employs equilibration between gas and liquid and a thermal conductivity detection gas chromatography for analysis. This device overcomes interferences by chromatographic separation but does not have sufficient sensitivity for the low-concentration applications contemplated by this invention.
Thus, to the best of applicant's knowledge no practical device capable of detecting concentrations on the order of 0.1 nM dissolved hydrogen exists with the sole exception of the reduction gas analyzer which is expensive and must be used in combination with the bubble strip method which is difficult to use.